Development of a truly effective anti-cocaine medication has been very challenging, particularly for treatment of cocaine overdose. There is still no FDA-approved anti-cocaine medication. Enhancing cocaine metabolism by administration of butyrylcholinesterase (BChE) has been recognized as a promising treatment strategy for cocaine abuse. However, the catalytic activity of this plasma enzyme is low against the naturally occurring (-) - cocaine. Our recent integrated computational-experimental effort has led to discovery of high-activity mutants of human BChE, known as cocaine hydrolases (CocHs), with >1,000-fold improved catalytic efficiency against cocaine compared to wild-type BChE. In vivo evidences indicate that our discovered CocHs are promising candidates for development of an anti-cocaine medication, especially for the overdose treatment. In this proposed project, we focus on the selection and optimization of the most promising CocH as a novel therapeutic candidate for cocaine overdose treatment through a combined use of various in silico, in vitro, and in vivo approaches. The specific aims are: (1) To prepare and characterize the discovered CocHs in vitro for their catalytic activity and stability; (2) To characterize the CocHs in vivo for their potency, biological/circulatory half-lives, and immunogenicity by using the CocH materials prepared in Aim 1; (3) To design, prepare, and characterize new CocH entities that have not only a high in vivo potency, but also a higher thermal stability and a longer circulatory half-life without immunogenicity. Accomplishment of this proposed investigation will result in the identification and development of the most promising CocH entity that has a high in vivo potency in the protective and rescuing effects, a high stability, and a sufficiently long biological half-life without immunogenicity. The CocH entity optimized in this investigation is expected to be highly effective and safe as an exogenous enzyme for cocaine overdose treatment in humans.